Electrochemical and photochemical electrodes and their use

ABSTRACT

Electrodes carrying FAD-dependent enzymes on their surface are enclosed. The FAD is modified to include a functional group or moiety which affects the properties of the enzyme. The functional group or moiety can be a binding moiety through which the FAD-enzyme complex is immobilized onto the electrode; it can be an electron mediator group for transferring electrons between the electrode and the FAD; or it can be a photoisomerizable group which can undergo photoisomerization which yields a change in the rate of the electrically induced catalytic activity of the enzyme. The electrodes can be used in electrochemical systems for deforming analytes in liquid medium or for recordal of optical signals.

FIELD OF THE INVENTION

The present invention is generally in the field of bioelectronics andconcerns electrically conducting solid matrices (to be referred toherein as “electrodes”) carrying redox enzymes such that an electriccharge can flow between the surface of the electrode and the enzymesrendering them catalytically active. Also provided by the invention is aprocess for the preparation of the electrodes as well as devices,systems and methods making use of such electrodes. In accordance withone embodiment, the invention is applied for the determination of thepresence and optionally the concentration of an analyte in a liquidmedium. In accordance with another embodiment, the immobilized enzymescan be switched by light into two distinct biocatalytic states thusallowing the transduction and amplification of recorded optical signalsthus fulfilling “read” and “write” functions, rendering such electrodesuseful in optical information storage and processing.

PRIOR ART

The prior art believed to be relevant as a background to the presentinvention consists of the following:

1. Degani, Y., Heller, A., J. Am. Chem. Soc., 110:2615, 1988.

2. Willner, I., Katz, E., Riklin, A., Kasher R., J. Am. Chem. Soc.,114:10965, 1992.

3. Willner et al., U.S. Pat. No. 5,443,701.

4. Lion-Dagan, M . Katz, E., Willner, I., J. Amer. Chem. Soc., 116791 3,1994.

5. Willner, I., Lion-Dagan, M., Marx-Tibbon, S., Katz, E., J. Amer.Chem. Soc., 117:6581, 1995.

6. Willner, I. and Rubin, S., Angen. Chem. Int. Ed. Engl. 35: 367 ,1996.

7. Willner, I., Riklin, A., Shoham, B., Rivenson, D., Katz, E., Adv.Mater., 5:912, 1993.

8. Massey, V., Hemmerich, P., in Flavins and Flavoproteins, V. Massey &C. H. Williams (Eds.), Elsevier, Amsterdam, 83-96, 1982.

9. Walsh, C., Fisher, J., Spencer, R., Graham, D. W., Ashton, W. T.,Brown, J. E., Brown, R. D., Rogers, E. F., Biochemistry, 78:1942, 1978.

10. Bückmann, A. F., Erdmann, H., Pietzch, M., Hall, J. M., Bannister,J. V. in K. Kuneoyagi (Ed.), Flavins and Flavoproteins, Gruyter, Berlin,p. 597, 1994.

11. Riklin, A., Katz, E., Willner, I., Stocker, A., Bückmann, A.F.,Nature, 376:672, 1995.

12. Willner, I., Liondagan, M., Marxtibbon S., Katz, E., J. Amer. Chem.Soc., 117: 6581, 1995

13. Namba, K., Suzuki, S., Bull. Chem. Soc. Jpn., 48:1323, 1975.

14. Katz, E., Schlereth, D. D., Schmidt, H. L., J. Electroanal. Chem.,367:59, 1994.

BACKGROUND OF THE INVENTION

Covalent coupling of redox active groups (ferrocene, bipyridinium, etc.)to amino acid residues of redox enzymes produces biocatalysts thatelectrically communicate with electrodes electrically “wired”enzymes⁽¹⁻³⁾. Enzymes modified by photoisomerizable groups (e.g.nitrospiropyran/nitromerocyanine) show different enzymatic activitiesfor the different light-induced generated photoisomer states^((4,5)).The use of photoswitchable biocatalysts as active matrices for opticalrecordings and optobioelectric devices was recently reviewed⁽⁶⁾.

Electrically-wired enzymes were employed for the determination ofanalytes in electrochemical cells by the attachment of theelectrobiocatalyse to electrodes^((3,7)). In all of the describedsystems, the functional electroactive or photoactive units are randomlydistributed around the protein. The effectiveness of electrical contactbetween the enzyme redox-center and the electrode is limited. As aresult, the rate of electron transfer between the enzyme redox center isrelatively slow. This results in competitive electron transfer reactionswith co-substrates (e.g. oxygen) or interfering substrates (e.g.oxidation of uric acid or ascorbic acid). As a result the magnitude ofthe resulting currents that assay the respective analytes are moderatelylow and the analysis had to be performed in an oxygen free environment.Special care had to be made to eliminate any interfering reagents fromthe analysis medium.

For many enzymes (e.g. flavoenzymes) the FAD-cofactor can be removedfrom the native protein to yield the unfolded apo-protein which can bereconstituted back with the natural cofactor or chemically modified FADcofactors to yield the bioactive enzyme⁽⁸⁻¹⁰⁾. The reconstitution ofapo-flavoenzymes with a FAD-cofactor bound to an electron mediator groupgenerated an “electro enzynze” that exhibited electrical contact withelectrode surfaces. Mediated electron transfer activates thereconstituted enzymes for the electrocatalytic oxidation of theirsubstrates⁽¹¹⁾.

Enzyme-electrodes for electrochemical determination of an analyte canoperate as non-invasive or invasive analytical devices. For invasiveanalyses the electrodes must be constructed of bio compatiblenon-hazardous substances, and the electrodes must be fabricated as thinneedles to exclude pain upon invasive penetration. The low surface areaof the electrodes must be compensated by a high electrical activity ofthe sensing biocatalysts to yield measurable current responses.

The functions of enzymes modified by randomly substitutedphotoisomerizable units are only incompletely switched by external lightsignals. The perturbation structure of the protein environment of theactive redox center of enzymes is only partially affected by remotephotoisomerizable units. This yields only to partial, incomplete,deactivation of the photoisomerizable enzyme⁽¹²⁾.

GENERAL DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide an electrochemicalmethod and system for the determination of the presence and optionallythe concentration of an analyte in a liquid medium.

It is furthermore an object of the invention to provide electrodes foruse in such method and system. It is particularly an object of theinvention to provide such electrodes comprising a solid, electricallyconducting matrix carrying immobilized enzymes such that electric chargeand flow between the electrode to the enzymes renders the enzymecatalytically active whereby they catalyze a reaction in which theanalyte to be assayed is converted into a product.

It is furthermore an object of the invention to provide such electrodeswith high and efficient electron transport between the electrode and theenzymes such that the electrode is essentially insensitive to thepresence of otherwise interfering redox reagents, i.e. there is aminimum of non-specific redox reactions.

It is furthermore an object of the invention to provideenzyme-electrodes where the entities immobilized on the electrodes arenon toxic and non immunogenic enabling the use of the electrode ininvasive analysis.

It is furthermore an object of the invention to provideenzyme-electrodes with photoswitchable enzymes immobilized on theelectrode surface, for use in the recordal of optical signals andtransduction of recorded optical signals.

It is another object of the invention to provide uses of the electrodesof the invention as well as processes for their preparations.

Other objects of the invention will be clarified from the descriptionbelow.

The present invention has two aspects: one aspect, to be referred toherein as the “first aspect” in which the electrode is useful for thedetermination of the presence and optionally the concentration of ananalyte in a liquid medium; and another aspect, to be referred to hereinas the “second aspect”, in which the electrical response of theelectrode is photoregulated (i.e. the degree of electrical response iscontrollable by irradiation of light at a specific wavelength) allowingthe use of the electrode in recordal of optical signals and theelectrical transduction of recorded optical signals. Both aspects of thepresent invention share a common denominator in that the electrodescarry immobilized enzymes, and in that the enzymes have functionalizedcofactors, i.e., cofactors modified by the addition of a functionalgroup or moiety (such enzymes to be referred to at times as“functionalized enzymzes”).

In accordance with the invention a functionalized enzyme may be obtainedby reconstituting an apo-enzyme (an enzyme without its cofactor) with aFAD modified by the addition of a functional group or moiety(“functionalized FAD”).

In accordance with one embodiment the functional group or moiety is abinding moiety capable of chemical association with, attachment to orbeing chemically sorbed onto the surface of the electrode. In accordancewith another embodiment, the functional group or moiety is an electronmediator group which is a group capable of reversibly changing its redoxstate and transfer electrons to and from the FAD. In accordance with afurther embodiment the functionalized group is a photoisomerizable groupwhich can change its isomerization state upon photostimulation. It ispossible also in accordance with other embodiments of the invention forthe functionalized FAD to have more than one functional group or moiety,e.g. a binding moiety and an electron mediator group, or a bindingmoiety and a photoisomerizable group, etc.

In the case of a functionalized FAD having an electron mediator group,and particularly such wherein the functionalized FAD has both a bindingmoiety and an electron mediator group, there is a highly efficientelectron transfer between the electrode and the FAD, yielding enzymeturnover rate which approaches maximal theoretical considerations. Suchan electrode which is useful particularly in accordance with the firstaspect of the invention, gives rise to a very high electrical responseto a change in analyte concentration. Furthermore, the high turnoverrate renders the electrode essentially insensitive to interfering agentssuch as non-specifically oxidizing or reducing agents, e.g. oxygen,ascorbic acid, uric acid, etc.

In accordance with the first aspect of the invention, the functionalizedFAD preferably comprises an electron mediator group. It should be notedthat where the functionalized FAD in the functionalized enzyme used inthe first aspect does not comprise an electron mediator group, there isan electron mediator group which may be freely tumbling in solution orindependently immobilized on the surface of the electrode, side by sidewith the modified FAD.

In accordance with the second aspect of the invention, wherein thefunctionalized FAD has a photoisomerizable group, enzymes have twocatalytic states representing “ON” and “OFF” states. This allows the“writing” of a photo event on the surface of the electrode which is then“memorized” by the electrode by means of the induced photoisomerizablestate of the functionalized cofactor, and this state can then be “read”by the electrode by measuring a change in the electrical response.

In the method and system of the invention, the changes in the analyte'sconcentration in the case of the first aspect or a change in thephotoisomerization state in the case of the second aspect gives rise toa change in the electrical response. The term “electrical response”which is used herein denotes the current-voltage behavior of anelectrode, e.g. the current response or the flow of charge of anelectrode under a certain applied potential, etc. The electricalresponse may be determined by measuring current or charge flow, underalternating current or direct current conditions.

In the following the term “determine” or “determination” will be used todenote both determination of only the presence or determination of boththe presence and concentration of an analyte in a liquid medium.

In the following, use will also be made of the term “reconstitution”referring to the joining together of an apo-enzyme (enzyme without itscofactor) with a cofactor to obtain a functionalized enzyme. Inaccordance with the invention the reconstitution of the enzyme isperformed with a synthetic, functionalized FAD-cofactor. The term“reconstituted functionalized enzyme” will be used to denote an enzymeobtained by reconstitution of an apo-enzyme with a functionalizedcofactor.

The functionalized enzyme reconstituted with a functionalized FAD willhave properties which will be influenced by the type of the FADmodification. For example, a functionalized enzyme having a FADcomprising a linking group with a binding moiety and having an electronmediator group will be electrobiocatalytically active and capable ofdirectly receiving electrons from or transferring electrons to theelectrode (depending on whether it catalyzes in a reduction or oxidationpathway, respectively), without the need for a separate electronmediator group. Such a functionalized enzyme exhibits a highly efficientelectrical contact with the electrode with an enzyme turnoverapproaching maximal theoretical consideration. A functionalized enzymewith a functionalized FAD having a photoisomerizable group will havedifferent electrobiocatalytic properties, depending on the isomerizationstate of the photoisomerizable group; the catalytic properties of theenzyme can thus be controlled by light.

In accordance with the teaching of the invention there is thus providedan electrode carrying FAD-dependent enzymes on its surface, the enzymeshaving a functionalized FAD, being an FAD modified by the addition of afunctional group or moiety, being one or more of the group consistingof:

(a) a binding moiety which can chemically associate with, attach to orchemically sorb onto the electrode, the enzyme being immobilized on theelectrode by binding of the binding group to the electrode's surface;

(b) an electron mediator group which can transfer electrons between thesurface of the electrode and the FAD; and

(c) a photoisomerizable group which can change from one isomerizationstate to another by exposure to light of a first wavelength, suchphotoisomerization either increases or decreases the electricallyinduced catalytic activity.

Preferred electrodes for use in accordance with the first aspect of theinvention are such wherein the functionalized FAD comprises both abinding moiety and an electron mediator group. Typically the electronmediator group will be sandwiched between the binding moiety and theremainder of the functionalized FAD thus allowing efficient and rapidelectron transfer between the surface of the electrode and the FAD.

In electrodes for use in accordance with the second aspect of theinvention the functionalized enzymes may at times be bound to theelectrode by means of a group linked to a surface residue of the proteinat its one end and having a binding moiety at its other end.Alternatively, the functionalized FAD may comprise both aphotoisomerizable group and a binding moiety bound to the electrode.

The present invention also provides a process for preparing an electrodehaving FAD-dependent redox enzymes immobilized thereon, the processcomprising:

(a) preparing apo-enzymes by treating an FAD-dependent enzyme so as toremove the FAD-cofactor therefrom;

(b) preparing a functionalized FAD by covalent binding to a bindingmoiety capable of chemical association with, attachment to or a chemicalsorption to the surface of the electrode;

(c) reacting the functionalized FAD with the electrode under conditionssuch that the modified FAD becomes immobilized onto the electrodethrough chemical association, attachment or sorption of the bindingmoiety onto the surface of the electrode; and

(d) reacting the electrode obtained in (c) with the apo-enzyme underconditions in which the apo-enzyme combines with the modified FAD toyield functional immobilized enzymes.

As will be appreciated, in the above process, steps (a) and (b) can bereversed. Furthermore, it is at times possible to first combine theapo-enzyme with the modified FAD and only then immobilizing the entirecomplex onto the surface of the electrode.

Where the functionalized FAD comprises other functional groups, i.e. anelectron mediator group or a photoisomerizable group, these may be, apriori, included in the modified FAD prior to its immobilization ontothe electrode, or may be added to the modified FAD after immobilization.A preferred immobilization scheme for preparing an electrode for use inaccordance with the first aspect, comprises:

(a) treating an electrode to obtain a monolayer comprising an electronmediator group, the electron mediator group having a binding moietywhich is capable of chemical association with, attachment to or chemicalsorption to the surface of the electrode, the treatment comprisingbinding of the binding moiety onto the surface of the electrode;

(b) reacting the electrode obtained in (a) with an FAD such that the FADbecomes immobilized onto the electrode through chemical attachment tothe electron mediator group;

(c) reacting the electrode obtained in (b) with apo-enzyme underconditions in which the apo-enzyme combines with the FAD component ofthe modified FAD.

The present invention further provides, by another of its facets, anelectrochemical system for determining the presence of an analyte liquidmedium, the system comprising:

(a) an electrode carrying on its surface FAD-dependent enzymes, theenzymes being capable of catalyzing a redox reaction in which an analyteis converted into a product, the enzymes comprising a functionalized FADhaving a binding moiety which is chemically associated with, attached toor chemically sorbed onto the surface of the electrode;

(b) an electron mediator group which can transfer electrodes between thesurface of the electrode and the FAD, the electron mediator group either

(ba) forming part of or being covalently bound to the functionalizedFAD,

(bb) being independently immobilized onto the surface of the electrode,

(bc) being covalently bound to the enzyme, or

(bd) being freely tumbling (i.e. being non immobilized) in a mediumsurrounding the electrode; and

(c) an electrical circuitry for charging the electrode and measuring theelectrical response.

As will be appreciated, the analyte specificity of the system isdetermined by the type of the immobilized enzyme.

The present invention further provides a method for determining thepresence of an analyte in a liquid medium, the method comprising:

(a) providing an electrochemical system as defined above;

(b) introducing a sample of said liquid medium into the electrochemicalcell of the system;

(c) charging the electrode and measuring the electrical response, achange in the electrical response as compared to an electrical responseunder the same condition in a control medium which does not comprise theanalyte, indicating the presence of the analyte in the system.

Electrodes in accordance with the first aspect of the invention exhibithigh turnover rates which approaches theoretical concentrations and arethus essentially insensitive to various non-specific oxidizing orreducing agents such as oxygen, etc. This is particularly the case inelectrodes of the invention where the electron mediator group forms partof or is covalently attached to the functionalized FAD. Such electrodesare thus suitable for performing measurement in a non protectedenvironment. e.g., measurement performed in vivo. A particular exampleis an electrode in the form of a needle which can be inserted into ablood vein and continuously measure a desired parameter, e.g. glucoselevel. All the entities of the surface of the electrodes, i.e. theenzymes, may be made to be identical to such normally present within thebody and accordingly there will typically be no immune response or anytoxic effect, which may otherwise result from a continuous exposure to aforeign entity.

Electrodes and systems for continuous in vivo measurement of variousparameters, are particularly preferred in accordance with the firstaspect of the invention.

Enzymes which can be used in accordance with the invention includeglucose oxidase (GOD), in which case the analyte will be glucose;D-aminoacid oxidase (DAAO), in which case the analyte is a D-aminoacid(e.g. D-alanine); lactate oxidase (LacOx), in which case the analyte islactic acid; glutathione reductase (GR), in which case the analyte isoxidized glutathione; and many other flavoenzymes.

In accordance with a second aspect of the invention there is provided anelectro chemical 'system for the recordal of optical signals having afirst wavelength and the electrical transduction of the recordedsignals, the system having an electrochemical cell comprising:

(a) an electrode carrying immobilized FAD-dependent redox enzymes, theenzyme:

(aa) having a functionalized FAD comprising a photoisomerizable groupwhich changes its isomerization state from a first to a second stateupon photostimulation of light of the first wavelength, a change in theisomerization state giving rise to a change in the rate of catalyticactivity of the redox enzyme,

(ab) being immobilized onto the surface of the electrode through alinking group which either

(aba) forms part of or being covalently bound to the functionalized FAD,or

(abb) is covalently bound to an external moiety on the surface of theenzyme;

(b) an electron mediator group which can transfer electrons between theelectrode and the FAD, the electron mediator group being either

(ba) freely tumbling in the medium surrounding the electrode,

(bb) independently immobilized onto the surface of the electrode,

(bc) covalently bound to the enzyme, or

(bd) covalently bound to or forming part of the modified FAD;

(c) a substrate for the catalytic activity of the enzyme; and

(d) an electric circuitry for charging the electrode and measuring theelectrical response.

Preferably, the photoisomerizable group can be isomerized reversibly byexposure to light to different wavelength regions. Thus, lightirradiation at a first wavelength will change the isomerization statefrom a first state to a second state whereas light of a secondwavelength will change the isomerization state between the second stateto the first state. Accordingly, the system may comprise a light sourceirradiating light at the second wavelength for changing theisomerization state from the second back to the first state. Thus, thesystem will record light events at a first wavelength and can then bereset by the second wavelength emitted from the system's light source.

The present invention further provides, in accordance with the secondaspect, a method for recordal of optical signals having a firstwavelength and electrical transduction of the recorded optical signals,the method comprising:

(a) providing an electro chemical system as defined above;

(b) exposing the electrode to a light source;

(c) charging the electrode and measuring the electrical response,changing the electrical response indicating exposure to light havingsaid first wavelength.

The present invention still further provides a process for preparingelectrodes for use in accordance with the second aspect of theinvention, the process comprising:

(a) preparing apo-enzyme by treating a FAD-dependent enzyme so as toremove the FAD therefrom;

(b) preparing a modified FAD by covalent binding of a group capable ofattachment or binding to a photoisomerizable group;

(c) reacting the modified FAD with the photoisomerizable group to yielda photoisomerizable FAD;

(d) combining the apo-enzyme with the photoisomerizable FAD to yield areconstituted photoisomerizable redox enzyme; and

(e) providing an electrode carrying linking groups immobilized thereonand reacting the reconstituted enzymes with the electrodes such that theenzymes become covalently bound to the linking group.

Another process to prepare an electrode in accordance with a secondaspect, comprises:

(a) preparing apo-enzyme by treating a FAD-dependent enzyme so as toremove the FAD therefrom;

(b) preparing a modified FAD by covalent binding of a group capable ofattachment or binding to a photoisomerizable group;

(c) reacting the electrode with a linking group having a binding moietycapable of association, chemical binding or sorption to the electrodeand having a functional unit capable of binding to a photoisomerizablegroup, the reaction being under condition so that said binding moietyassociates, chemically binds or sorbs with the surface of the electrode;

(d) reacting the electrode obtained in (c) with a photoisomerizablegroup;

(e) reacting the electrode obtained in (d) with the modified FADobtained in (b), such as to obtain a monolayer comprising immobilizedphotoisomerizable FAD moieties on the electrode; and

(f) reacting the apo-enzymes with the electrode obtained in (e) undercondition whereby the enzyme is reconstituted on the surface of theelectrode thus yielding photo active redox enzymes immobilized on theelectrode.

Enzymes which can be used in accordance with the second aspect of theinvention include those mentioned above in connection with the firstaspect.

A linking group which can be utilized in accordance with the presentinvention to immobilize an FAD onto the surface of an electrode, mayhave the following general formula (I):

Z—R¹—Q  (I)

wherein:

Z is a binding moiety in case where the electrode is made of gold,platinum or silver, represents a sulphur-containing moiety which iscapable of chemical association with, attachment to or chemisorptiononto said metal; and in case where the electrode is made of glass,represent methoxy or alkoxy silane residues which are capable ofchemical association, attachment to or chemisorption onto said glass;

R¹ represents a connecting group;

Q is a functional group which is capable of forming a covalent bond witha moiety in the catalytic peptide or in the porphyrin group.

Z, where the electrode material is a metal, may for example be a sulphuratom obtained from a thiol group, a disulfide group, a sulphonate group,or a sulphate group.

R¹ may be a covalent bond or may be a peptide or polypeptide or may beselected from a very wide variety of suitable groups such as alkylene,alkenylene, alkynylene phenyl containing chains, and many others.

Particular examples of R¹ are a chemical bond or a group having thefollowing formulae (IIa), (IIb), (IIc) or (IId)

wherein

R² or R³ may be the same or different and represent straight or branchalkylene, alkenylene, alkynylene having 1-16 carbon atoms or represent acovalent bond,

A and B may be the same or different and represent 0 or S,

Ph is a phenyl group which is optionally substituted, e.g. by one ormore members selected from the group consisting of SO³ ⁻ or alkylgroups.

Q may for example be an amine group, capable of binding to a carboxylresidue; a carboxyl group, capable of binding to an amine residue; anisocyanate or isothiocyanate group or an acyl group capable of bindingto an amine residue; or a halide group capable of binding to hydroxyresidues of the polypeptide. Particular examples are the groups—NH₂—COOH; —N═C═S; N═C═O; or an acyl group having the formula—R^(a)—CO—G wherein G is hydrogen, a halogen such as Cl, or is OH,OR^(b), a

group or a

group; R^(a) and R^(b) being, independently a C₁-C₁₂ alkyl or alkenyl ora phenyl containing chain which is optionally substituted, e.g. byhalogen.

Particular examples of such a linking group are those of the followingformulae (III)-(IX):

wherein n is an integer between 1-6.

Linking the FAD with an electro mediator group or a photoisomerizablegroup, as well as linking of electron mediators directly onto theenzyme, may be achieved by means of a connecting group having thefollowing formula (X):

Z—F—R¹—Q  (X)

wherein R¹ has the same meaning as indicated above and Q¹ and Q² haveindependently one of the meanings given above for Q.

Examples of electron-mediator groups which can be used in accordancewith the invention are errocene, pyrroloquinoline quinone, quinone,N,N′-dialkyl-4,4′-bipyridinium salts and many others.

The linking group may at times comprise also another functional group,such as an electron mediator group or a photoisomerizable group. Alinking group in accordance with such embodiments may have the followinggeneral formula (XI)

Q¹—R¹—Q²  (XI)

wherein Z, R¹ and Q have the meaning given above, and F is thefunctional group.

Examples of photoisomerizable groups that can be used in accordance withthe invention are nitrospiropyran, azobenzene, thiophene fulgide andmany other compounds being photoisomerized from one state to the otherstate and back by irradiation by light of two different wavelengthregions.

The present invention will now be further illustrated in the followingspecific embodiments and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of N⁶-(2-aminoethyl)-FAD.

FIGS. 2A and 2B show the last synthetic step in preparation ofFAD-ferrocene diad.

FIG. 3A shows the last synthetic step in preparation of FAD-spiropyrandiad (FAD-SP).

FIG. 3B shows the two photoisomeric states of the FAD-SP.

FIG. 4 shows the preparation of glucose oxidase apo-enzyme and itsreconstitution with FAD-ferrocene diad.

FIG. 5A shows the preparation of glucose oxidase apo-enzyme and itsreconstitution with FAD-spiropyran diad and the photoisomerization ofthe reconstituted enzyme;

FIG. 5B shows the two photoisomeric states of the modified FAD.

FIG. 6 shows the scheme of immobilization of GOD reconstituted withFAD-spiropyran diad, onto a gold electrode to form a GOD monolayer.

FIG. 7 shows the scheme of modification of a gold electrode with an FADmonolayer and the bioclectrocatalytic glucose oxidation using theseenzyme electrodes;

FIG. 7A shows an electrode in accordance with the first embodiment ofthe invention with electron mediator group freely tumbling in themedium;

FIG. 7B shows an enzyme in accordance with the combined first and secondembodiments with an electron mediator group (PQQ) covalently bound tothe FAD group.

FIG. 8 shows the structure of pyrroloquinoline quinone (PQQ).

FIG. 9 shows cyclic voltammograms: (a) FAD-ferrocene (see FIG. 4)adsorbed onto an Au working electrode (from a 1·10⁻⁵ M stock solution);(b) FAD-ferrocene reconstituted GOD in solution (1.75 mg mL⁻¹) (see FIG.12) using a cystamine monolayer modified Au electrode. Background: 0.1 Mphosphate buffer, pH 7.3, under argon. Potential scan rate, 1.5 V s⁻¹.

FIG. 10 shows cyclic voltammograms of FAD-ferrocene-reconstituted GOD insolution (1.75 mg mL⁻¹) and different concentrations of glucose: (a) 0mM, (b) 1 mM, (c) 3 Mm, (d) 20.5 mM. All experiments were performed in0.1 M phosphate buffer, pH 7.3, at 35° C.; under argon, using acystamine modified Au electrode. Potential scan rate, 2 mV s⁻¹.

FIG. 11 shows the peak anodic currents at different glucoseconcentrations in an experiment such as that shown in FIG. 10.

FIG. 12 shows the bioelectrocatalytic oxidation scheme of glucose usingFAD-ferrocene reconstituted GOD (“electroenzyme”).

FIG. 13 shows cyclic voltammograms of FAD-ferrocene reconstitutedD-aminoacid oxidase (DAAO) in solution (0.38 mg/ml) in the presence ofD-alanine: (a) 0 mM, (b) 2 mM, (c) 9 mM. Experiment carried out in 0.1 Mpyrophosphate buffer, pH 8.5; 25° C.; under argon. Potential scan rate,2 mV s⁻¹.

FIG. 14 shows cyclic voltammograms of the bioelectrocatalyzed oxidationof glucose, 5·10⁻² M, and ferrocene carboxylic acid, 5-10⁻⁴ M, in thepresence of: (a) FAD-SP-GOD, 0.46 mg/ml, (b) FAD-MRH⁺-GOD, 0.46 mg/ml.All experiments were recorded in a 0.1 M phosphate buffer, pH 7.0, 37°C., under argon. Potential scan rate, 5 mV s⁻¹.

FIG. 15 shows bioelectrocatalytic oxidation schemes of glucose usingFAD-SP reconstituted GOD (photoswitchable enzyme).

FIG. 16 shows Lineweaver-Burk plot for the saturation-current curves:

(i) FAD-SP-GOD (); (ii) FAD-MRH+-GOD (∘).

FIG. 17 shows the different isomeric forms providing “ON” (left) and“OFF” (right) states for the monolayer immobilized FAD-SP-GOD.

FIG. 18 shows cyclic voltammograms of different electroenzymaticactivities for glucose oxidation by monolayer immobilized FAD-SP-GODbeing in different isomeric states. Glucose concentration, 50 mM.Potential scan rate, 5 mV s⁻¹.

FIG. 19 shows cyclic amperometric transduction of optical signalsrecorded by the reconstituted photoisomerizable GOD monolayerimmobilized onto a gold electrode; (∘) SP-state, (□) MRH⁺-state.

FIG. 20 shows cyclic voltammograms of GOD reconstituted onto aFAD-modified monolayer Au electrode: (a) background electrolyte solutiononly; (b) in the presence of ferrocene carboxylic acid, 4 10⁻⁴ M; (c)with ferrocene carboxylic acid and added glucose, 5 10⁻²M. Allexperiments were recorded under argon in 0.01 M phosphate buffer and 0.1sodium sulfate, pH 7.0, 35° C., scan rate 5 mV s⁻¹.

FIG. 21 shows cyclic voltammograms of the PQQ-FAD diad monolayer Auelectrode (a) and of the PQQ-FAD diad monolayer after reconstitutionwith apo-GOD (b). All experiments were recorded under argon in 0.01 Mphosphate buffer and 0.1 sodium sulfate, pH 7.0, 25° C., scan rate 50 mVs⁻¹.

FIG. 22 shows cyclic voltammograms of GOD reconstituted onto the PQQ-FADdiad monolayer electrode; without glucose (a) and in the presence of 80mM glucose (b). All experiments recorded under argon in 0.01 M phosphatebuffer and 0.1 sodium sulfate, pH 7.0, 35° C., scan rate, 5 mV s⁻¹.

FIG. 23 shows amperometric responses of GOD reconstituted onto thePQQ-FAD diad monolayer electrode at different glucose concentration.Currents determined by chronoamperometry at final potential +0.2 V, 35°C..

FIG. 24 shows an amperometric response produced by GOD reconstitutedonto a PQQ-FAD monolayer: (a) in the absence of glucose; and (b) in thepresence of 50 mM glucose, in the absence of O₂; (c) in the presence of50 mM glucose in a solution saturated with air; (d) in the presence of50 mM glucose, 0.1 mM ascorbic acid in a solution saturated with air.Currents were determined by chronoamperometry at a final potential of0.0V vs. SCE. The electrolyte consisted of 0.01 M phosphate buffer and0.1 M sodium sulfate, pH 7.0, with the measuring temperature being35±0.5° C.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The following specific embodiments are intended to illustrate theinvention and shall be construed as limiting its scope. The artisan willno doubt appreciate that these specific embodiments are an example ofthe full scope of the invention as defined above.

EXAMPLES

1. Chemical Synthetic Steps and Biochemical Preparations

1.1 Last Synthetic Step in Preparation of FAD-Ferrocene Derivative

Amino derivatized FAD, N⁶-(20aminoethyl)-FAD, (FIG. 1) was synthesizedaccording to the results published recently procedure⁽¹⁰⁾.N-(2-methylferrocene) caproic acid was synthesized as recentlydescribed⁽⁷⁾. N⁶-(2-aminoethyl)-FAD (10 mg, 1.1·10⁻⁵ mol) was reactedwith N-(2-methyl-ferrocene) caproic acid (18 mg, 5.5·10⁻⁵ mol) in thepresence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC,Aldrich; 11.6 mg, 5.5·10⁻⁵ mol) as a coupling reagent andN-hydroxy-3-sulfosuccinimide sodium salt (NSI, Aldrich; 13.2 mg,5.5·10⁻⁵ mol) as a promoter (FIG. 2). The coupling reaction was done in1 ml of 0.1 M HEPES buffer, pH 7.4 for 3 h at room temperature. Theproduct was purified on Sepadex G10 column using water as eluent. Theseparation from the non-reacted original compounds was performed by athin layer chromatography (TLC) R_(g)=0.49, 0.69, 0.93 for amino-FAD,Fc-COOH and the diad FAD-Fc, respectively). The structure of FAD-Fc wasconfirmed by H¹-NMR spectrum.

1.2 Last Synthetic Step in Preparation of FAD-Spiropyran derivative

Carboxylic derivative of spiropyran,1′-(β-carboxymethyl)-3′,3′-dimethyl-6-nitro-[2H-1]benzopyran-2,2′-indolin,SP-COOH, was synthesized as hitherto described (Namba and Suzuki, Bul.Chem. Soc. Jpn, 48:1323, 1975)⁽¹³⁾. The diad including the amino-FAD andthe photoisomerizable component (FAD-SP) was prepared similarly asdescribed above for FAD-Fc using SP-COOH as carboxylic component forcoupling with the amino-FAD (FIG. 3). The product was purified bypreparative TLC on SiO₂ plates using isopropanol:H₂O (7:3) as eluent.The nitrospiropyran-modified FAD reveals reversible photoisomerizableproperties. Illumination of FAD-SP, 360 nm<λ<380 nm (“hv” in FIG. 3),yields the nitromerocyanine-FAD isomer state, FAD-MRH⁺, exhibiting anabsorption band in the region of 320-560 nm that corresponds to theoverlapping bands of the MRHP⁺ (520 nm) and FAD (355 nm, 460 nm)chromophores. Irradiation of the MRH⁺-FAD solution, λ>475 nm (“hv₁” inFIG. 3) yields a yellow solution exhibiting the FAD absorption band atλ=360 nm, 447 nm and the characteristic SP-absorption in the UV region.The photoisomerization between the FAD-SP and FAD-MRH⁺ is reversible(FIG. 3).

1.3 Apo-Enzyme Preparations

Apo-glucose oxidase (apo-GOD) was prepared similarly as hithertodescribed (Morris and Buckler in Methods in Enzymology, Vol. 92, Part E,(J. J. Langone and V. Van Vunakis, Eds.), Academic Press, Inc. pp.415-417, 1983) by acidification of a glucose oxidase, GOD, solution(from Aspergillus nigger, E.C. 1.1.3.4) to pH=1.7, followed byseparation on a Sephadex G-25 column and further purification withcharcoal-dextran, and dialysis against 0.1 M phosphate buffer, pH 7.0,for 24 hours at 4° C..

Apo-protein derived from D-aminoacid oxidase (DAAO; from pig kidney,E.C. 1.4.3.3) was prepared following a similar procedure as hithertodescribed (Massey and Curti, J. Biol. Chem., 241:3417, 1966) as follows:The enzyme was dialyzed against a 0.1 M pyrophosphate buffer, pH 8,5,containing 1 M KBr and 3·10⁻³ MEDTA, followed by dialysis against a 0.1M pyrophosphate buffer, pH 8,5, and finally it was purified by the sameway as apo-GOD. Both apo-proteins do not show any enzymatic activity.

1.4 Reconstitution of Apo-Enzymes with Diads: FAD-FC and FAD-SP

The apo-GOD was reacted with FAD-Fc diad to generate the ferrocene-FADreconstituted glucose oxidase (FIG. 4). The reconstitution was done in 3ml stirred Na-phosphate buffer, 0.1 M, pH 7:0, containing 9.6 mg(5.16·10⁻⁵ mol) apo-GOD and 0.8 mg (6.7·10⁻⁴ mol) FAD-Fc for 4 h at roomtemperature followed by overnight incubation at 4° C.. The solution waspurified twice by filtration through a filter with a cut of 10,000 andthen dialyzed against 0.1 M Na-phosphate buffer, pH 7.0 for one day.

The loading of the reconstituted GOD was determined spectroscopically asone molecule of the FAD-Fc diad per the enzyme subunit. The activity ofthe reconstituted GOD was about 40% of that of the native GOD.

The apo-DAAO was reconstituted with the FAD-Fc diad and then purifiedsimilarily as described above for the apo-GOD. The loading of thereconstituted DAAO was about one molecule of the FAD-Fc diad per theenzyme. The activity of the reconstituted GOD was about 20% of that ofthe native GOD.

Reconstitution of the apo-GOD with the FAD-SP diad (FIG. 5) wasaccomplished by treatment of apo-GOD with the diad (molar ratio 1:10) in0.1 M phosphate buffer, pH 7.0 under vigorous shaking for 24 hours, 25°C.. The product was dialyzed against phosphate buffer, 0.1 M, pH 7.0,for 30 hours. The loading of the FAD-SP cofactor was about 1 per each ofthe enzyme subunits. Reconstituted GOD exhibits about 80% of theactivity of the native GOD. The reconstituted GOD exhibitsphotoisomerizable properties. Illumination of the GOD reconstituted withFAD-SP, 360 nm <λ<380 nm, yields the merocyanine isomeric state that canbe isomerized back using illumination λ>475 nm (FIG. 5).

2. Electrode Characterization and Electrochemical Set-Ups

Gold electrodes (0.5 mm diameter Au wire, having a geometrical area ofabout 0.2 cm²; roughness coefficient of about 1.2 or Au foil ofgeometrical area of about 0.4 cm²; roughness coefficient of about 15)were used for all modifications and measurements. The rough goldelectrode was obtained by treatment with liquid mercury and furtherdissolution of the amalgam layer in concentrated nitric acid (Katz etal., J. Electroanal. Chem., 367:59, 1994). A cyclic voltammogramrecorded in 0.5 M H₂SO₄ was used to determine the purity of theelectrode surface just before modification. The real electrode surfacearea and coefficients of roughness were estimated from the same cyclicvoltammogram by integrating the cathodic peak for the electrochemicalreduction of the oxide layer on the electrode surface (Woods in A. J.Bard (Ed.), Electroanalytical Chemistry, Dekker, New York, p. 1, 1978).

Electrochemical measurements were performed using a potentiostat (EG&G)VersaStat) connected to a personal computer (EG&G researchelectrochemistry software model 270/250). All the measurements werecarried out in a three-compartment electrochemical cell comprising thechemically modified electrode as a working electrode, a glassy carbonauxiliary electrode isolated by a glass frit and a saturated calomelelectrode (SCE) connected to the working volume with a Luggin capillary.All potential are reported with respect to this reference electrode.Argon bubbling was used to remove oxygen from the solutions in theelectrochemical cell. During the measurements the cell was thermostatedusing circulated water in a jacket around the cell (the temperature isindicated in each of the experimental examples below).

3. Electrode Modification

3.1 Pretreatment

To remove a previous organic layer and to regenerate a bare metalsurface, the electrode was treated with a boiling 2 M solution of KOHfor 1 h, then rinsed with water and stored in concentrates sulfuricacid. Immediately before modification, the electrode was rinsed withwater, soaked for 10 min in concentrated nitric acid and then rinsedagain with water.

3.2 Electrode Modification with Cystamine

A clean bare gold electrode was soaked in a solution of 0.02 M cystamine(2,2′-diaminodiethyldisulfide, Aldrich) in water for 2 h. The electrodewas then rinsed thoroughly with water to remove the unabsorbedcystamine.

3.3 Monolayer Immobilization of the Photoswitchable GOD Reconstitutedwith FAD-SP

A gold electrode was functionalized with active ester groups bychemisorption of dithio-bis-(succinimidylpropionate) (DSP, Aldrich)(Willner et al., 1992²; Katz, E., J. Electroanal. Chem., 191:257,(1990)) and then the electrode was treated with the GOD reconstitutedwith FAD-SP (4 mg per 1 ml of 0.01 M phosphate buffer, pH 7.0) for 1 h(FIG. 6). The enzyme electrode was rinsed with 0.1 M phosphate buffer,pH 7.0 and used immediately for electrochemical measurements.

3.4 Electrode Modification with a FAD Monolayer

A lipoic acid active ster monolayer was adsorbed (1 h, room temperature)onto a rough gold electrode from 10 mM solution in dimethylsulfoxide(DMSO). The resulting electrode was rinsed twice with DMSO and once withwater to remove physically adsorbed molecules. Then the modifiedelectrode was treated with 5 mM N⁶-(2-aminoethyl)-FAD solution in 0.1HEPES buffer, pH 7.3 for 1 h and finally rinsed several times with water(FIG. 7A).

3.5 Electrode Modification with a PQQ-FAD Monolayer

The electrode modification with pyrroloquinoline quinone, PQQ, (FIG. 8)was done as recently described⁽¹⁴⁾. A cystamine monolayer Au modifiedelectrode was soaked for 3 h in a 0.01 M HEPES buffer solution, pH 7.3,containing 1 mM PQQ (Sigma). Then the modified electrode was thoroughlyrinsed with water to remove from its surface uncoupled physicallyadsorbed PQQ molecules. The obtained PQQ-functionalized electrode wastreated with 5 mM N⁶-(2-aminoethyl)-FAD solution in 0.1 HEPES buffer, pH7.3, in the presence of 10 mM EDC for 1 h and then thoroughly rinsedagain with water (FIG. 7B).

3.6 Apo-GOD Reconstitution onto FAD or PQQ-FAD Functionalized ElectrodeSurfaces

Each FAD-functionalized electrode (FAD only and PQQ-FAD) was treatedwith an apo-GOD solution (about 3 mg/ml in phosphate buffer, 0.1 M, pH7.0) under vigorous shaking for 4 hours at room temperature and then for16 hours at 4° C.. Then the modified electrode was rinsed with the 0.1 Mphosphate buffer, pH 7.0 and used immediately for electrochemicalmeasurements (FIGS. 7A&B).

4. Results

4.1 Electroenzyme: GOD Reconstituted with FAD-FC

The FAD-Fc diad exhibits in an aqueous buffer solution twocharacteristic reversible waves at −0.50 and 0.35 V (vs SCE) (FIG. 9).

These waves correspond to the two-electron redox process of FAD and theone-electron redox reaction of the ferrocene, respectively. Theelectrochemical process shows strong adsorption of the FAD-Fc diad onthe unmodified Au electrode. The cyclic voltammogram of theFAD-Fc-reconstituted GOD shows only the reversible redox process of theferrocene unit, implying that the ferrocene component communicates withthe electrode where the enzyme-embedded FAD component lacks directelectrical communication with the electrode (FIG. 9).

A cystamine modified electrode was used for electrochemical measurementsof the reconstituted GOD to prevent the protein adsorption that couldresult in the enzyme denaturation. FIG. 10 shows the electrocatalyticanodic currents developed by the FAD-Fc-reconstituted GOD in thepresence of different concentrations of added glucose. The calibrationcurve, showing the anodic current at different concentrations, is givenin FIG. 11. The electrobiocatalyzed oxidation of glucose can be analyzedin terms of the Michaelis-Menten model (I_(max)=4 μA and K_(m)=2.9 mM,where I_(max) is the saturation current and K_(m) is theMichaelis-Menten constant). Taking into account the surface area androughness factor of the working electrode, this maximum current densitycorresponds to I_(max)=8.3 μA/cm². For comparison, native glucoseoxidase under comparable conditions in the presence of ferrocenecarboxylic acid yields the values K_(m)3.3 mM and I_(max)=6.3 μA/cm². Itcan thus be concluded that reconstitution of apo-GOD with theferrocene-modified-FAD yields a semi-synthetic electroenzyme exhibitingelectrical communication between the electrode and the biocatalystactive site (FIG. 12).

the reconstituted DAAO showed electrical communication with electrodesurface and electrocatalytic anodic currents were observed in thepresence of D-alanine as substrate. FIG. 13 shows the cyclicvoltammograms observed upon addition of different concentrations ofD-alanine to a solution that contains the FAD-Fc DAAO. The respectivecalibration curve was similarly analyzed in terms of theMichaelis-Menten model and the values I_(max)=3.96 μA/cm² and K_(m)=2.0mM were derived for the semi-synthetic electroactive DAAO. Similar tothe reconstituted GOD, this reconstituted enzyme thus also functions asan electroenzyme.

4.2 Reconstitution of Apo-Glucose Oxidase with aNitrospiropyran-Modified FAD Cofactor Yields a PhotoswitchableBiocatalyst

The apo-GOD reconstituted with photoisomerizable FAD-SP diad has nodirect non-mediated electrical communication with an electrode.Therefore, bioelectrocatalytic glucose oxidation using thisreconstituted enzyme was studied in the presence of diffusionally mobileelectron transfer mediators (ferrocene derivatives: ferrocenemonocarboxylic acid, ferrocene dicarboxylic acid and dimethylaminoethylferrocene). The reconstituted enzyme was applied in the solubilized formor immobilized as a monolayer (see 3.3). FIG. 14 shows the cyclicvoltammograms obtained upon electrobiocatalized oxidation of glucose inthe presence of ferrocene carboxylic acid as a diffusional electrontransfer mediator and the FAD-SP-reconstituted GOD. The electrocatalyticanodic current in the presence of the FAD-MRH⁺ reconstituted GOD isenhanced by about 25% as compared to the FAD-SP-reconstituted GOD,implying the higher activity of the FAD-MRH⁺-GOD. The anocid currentsdeveloped by the systems in the presence of glucose are different forboth isomeric states: SP and MRH⁺: the system is “ON” and “OFF” (FIG.15). The detailed Michaelis-Menten kinetic analysis of the biocatalyticperformances of FAD-SP-GOD and FAD-MRH⁺-GOD in the presence of differentconcentrations of ferrocene carboxylic acid is shown in FIG. 16. The twophotoisomer states of the enzyme reveal similar I_(max)=6.9·10⁻⁶ Avalues, where the K_(m) values of the two enzyme states differsubstantially (7.82 M⁻¹ and 2.57 M⁻¹ for FAD-SP-GOD and FAD-MRH⁺-GOD,respectively). These results imply that the electron transfer rate ofthe oxidation of the FAD cofactor by the ferrocenylium cation is ofsimilar effectiveness in the two photoisomer states of the reconstitutedGOD, but the interactions of the electron mediator with the protein toattain the appropriate configuration for electron transfer differ forboth isomers. For low concentrations of the electron transfer mediatorsthe difference in the biocatalytic activity was higher and thisdifference depended on the kind of the electron transfer mediator whichwas used. For example, the difference in the enzyme activities for SPand MRH+states was even higher if dimethylaminoethyl ferrocene was usedas an electron transfer mediator (results not shown).

The FAD-SP-reconstituted GOD was assembled as a monolayer and appliedfor biocatalytic glucose oxidation in the presence of diffusionallymobile electron transfer mediator. The monolayer can be transformed intotwo different isomeric states by light (FIG. 17). FIG. 18 shows cyclicvoltammograms for reversible activation and deactivation of themonolayer modified electrode for bioelectrocatalytic glucose oxidationby light. The activation (biocatalytic system comprising thereconstituted GOD and diffusional mediator is in “ON” state) anddeactivation (the system is in “OFF” state) can be reversibly repeatedmany times (FIG. 19).

4.3 Electrical Wiring of Glucose Oxidase by Reconstitution ofFAD-Modified Monolayers Assembled onto Au-Electrodes

The electrode modified with the FAD component only (see 3.4) was used toreconstitute the apo-GOD directly on the interface. The immobilized FADrevealed a characteristic reversible cyclic voltammogram, E° (atpH=7.0)=−0.50 V vs SCE. The interfacial electron transfer rate constantbetween the electrode and FAD unit was ca. 230 s⁻¹ and the surfaceconcentration of the FAC units on the electrode was ca. 3·10⁻¹¹ mol/cm²that corresponds to a non-densely packed monolayer coverage. However,the reconstituted GOD on the electrode did not show any biocatalyticactivity in the absence of electron transfer mediators. This resultsfrom the inability of electrons to transfer between the electrode andthe FAD units incorporated into the apo-protein. It should be noted thatthe distances between the electrode and the electrochemical centers ofthe FAD unity depends on the mobility of the FAD units. Free(non-incorporated into the protein) FAD units are immobilized throughlong flexible spacers, are mobile and can thus exchange electrons withthe electrode; but the FAD units incorporated already into the proteinlost this mobility, and have a long distance from the electrode surfaceand can thus not communicate electrically with the electrode. However,the biocatalytic activity can be achieved in the presence ofdiffusionally mobile electron transfer mediator (FIG. 20).

To improve the system described above, an electrode modified with tworedox components: PQQ and FAD was used (see 3.5). FIG. 21 (curve a)shows the cyclic voltammogram of the PQQ-FAD diad monolayer modifiedelectrode. It consists of two redox waves at E° (at pH=7.0)=−0.125 and−0.50 V vs SCE corresponding to the two-electron reduction of the PQQand FAD units, respectively. The surface density is ca. 3·10⁻¹¹ molcm{circumflex over ( )}P2 for each component of the monolayer. ThePQQ-FAD monolayer was further treated with apo-GOD to produce thereconstituted GOD directly on the modified interface. FIG. 21 (curve b)shows the cyclic voltammogram of the resulting electrode afterreconstitution. The redox wave characteristic of the FAD units decreasesdramatically, whereas the redox wave corresponding to the PQQ componentis not changed. This is consistent with the fact that the FAD componentembedded n the protein as a result of reconstitution, lacks electricalcommunication with the electrode surface. The small residual FAD waveobserved after reconstitution corresponds to free FAD units that stillcommunicate with the electrode. From the difference in the FAD wavebefore and after reconstitution the surface density of the GOD on theelectrode surface was estimated to be about 1.7·10⁻¹² mol/cm². Theenzyme reconstituted onto the PQQ-FAD diad monolayer reveals directelectrical communication with the electrode and is active in thebioelectrocatalyzed oxidation of glucose. FIG. 22 shows the cyclicvoltammograms of the PQQ-FAD reconstituted GOD monolayer electrode inthe absence (curve a) and presence (curve b) of glucose. A highelectrocatalytic anodic current is observed with glucose indicating thatthe reconstituted protein bioelectro-catalyzes the glucose oxidationvery efficiently. The PQQ component of the monolayer functions as anelectron transfer mediator between the FAD incorporated into the proteinand the electrode. The electrocatalytic anodic currents developed by thesystem are controlled by the glucose concentration in the range 5-80 mM(FIG. 23). Dioxygen affect the electron transfer from the reconstitutedGOD through PQQ to the electrode only slightly (for 80 mM glucose theanodic current decreased in the presence of oxygen only by about 5%)that is very unusual for glucose biosensors based on GOD (results notshown). Addition of 0.1 mM ascorbate (usual interfering component invivo) to the system containing 5 mM glucose did not affect theamperometric response of the electrode. These results suggest that thereconstituted PQQ-FAD-GOD monolayer exhibits efficient electricalcommunication with the electrode surface that competes with theinterfering paths.

4.4 Turnover Rate of GOD Reconstituted onto a PQQ-FAD Monolayer: Lack ofInterference from Oxidizing and Reducing Agents

The upper limit of the turnover rate of glucose oxidase at 25° C. isGOD±100 s⁻¹ (C. Bourdillon et al., J. Am. Chem. Soc., 115:12264 (1993))and the activation energy is 7.2 Kcal·mole⁻¹ (H. G. Eisenwiener,Naturwissenschaften, 56:563, (1969)). At the temperature employed in theelectrobiochemical measurements shown in FIG. 22 and FIG. 23, (35° C.)this translates to a limiting turnover rate of 900±150 s⁻¹ at 35° C. Thesurface coverage of the reconstituted enzyme on the electrode is1.7·10⁻² and using the theoretic turnover rate at 35° C., the maximumcurrent density that can be observed from the electrode is 290±60μA·cm⁻². FIG. 23 shows that at a glucose concentration of 80 mM, theobserved current is 1.9 mA (for an electrode with a surface area of 0.4cm² and roughness factor of Ca.20). This translates to an experimentalcurrent density corresponding to 300±100 μA·cm⁻², a value that is withinthe range of the limiting turnover rate of the enzyme. This is furthersupported by the fact that the current response is linear with glucoseconcentration, FIG. 23, indicating that the S current is controlled bythe diffusion of glucose to the active site and that the FAD sites existin their oxidized form at E≈0.2 Volt.

The essentially lack of interference from surrounding redox reagents isdemonstrated in FIG. 24. This is a result of the very high turnover rateof the enzyme which renders it essentially insensitive to non-specificinterferences. Thus, an electrode of this kind can be used forcontinuous measurement in an unprotected environment, e.g. in vivo.

What is claimed is:
 1. A process for preparing an electrode havingFAD-dependent redox enzymes immobilized thereon, the process comprising:(a) preparing apo-enzymes by treating an FAD-dependent enzyme so as toremove the FAD-cofactor therefrom; (b) preparing a functionalized FAD bycovalent binding to a binding moiety capable of chemical associationwith, attachment to or a chemical sorption to the surface of theelectrode; (c) reacting the functionalized FAD with an electrode underconditions such that the modified FAD becomes immobilized onto theelectrode through chemical association, attachment or sorption of thebinding moiety onto the surface of the electrode; (d) reacting theelectrode obtained in (c) with the apo-enzyme under conditions in whichthe apo-enzyme combines with the modified FAD to yield functionalimmobilized enzymes; and (e) reacting the electrode obtained in (d) withan enzyme substrate in the presence of a diffusional electron mediator.2. A process for preparing an electrode having FAD-dependent redoxenzymes immobilized thereon, the process comprising: (a) treating anelectrode to obtain a monolayer comprising an electron mediator group,the electron mediator group having a binding moiety which is capable ofchemical association with, attachment to or chemical sorption to thesurface of the electrode, the treatment comprising binding of thebinding moiety onto the surface of the electrode; (b) reacting theelectrode obtained in (a) with an FAD such that the FAD becomesimmobilized onto the electrode through chemical attachment to theelectron mediator group; (c) reacting the electrode obtained in (b) withapo-enzyme of an FAD-dependent enzyme under conditions in which theapo-enzyme combines with the FAD component of the immobilized FAD.
 3. Aprocess for preparing electrodes comprising immobilized enzymes with anassociated photoisomerizable group which can change its isomerizationstate by exposure to light at a certain wavelength, thereby changing therate of electrically induced catalytic activity of the enzyme, theprocess comprising: (a) preparing an apo-enzyme by treating aFAD-dependent enzyme so as to remove the FAD therefrom; (b) preparing amodified FAD by covalent binding of a group capable of attachment orbinding to a photoisomerizable group; (c) reacting the modified FAD withthe photoisomerizable group to yield a photoisomerizable FAD; (d)combining the apo-enzyme with the photoisomerizable FAD to yield areconstituted photoisomerizable redox enzyme; and (e) providing anelectrode carrying linking groups immobilized thereon and reacting thereconstituted enzymes with the electrodes such that the enzymes becomecovalently bound to the linking group.
 4. An electrochemical system fordetermining the presence of an analyte liquid medium, the systemcomprising: (a) an electrode carrying on its surface FAD-dependentenzymes, the enzymes being capable of catalyzing a redox reaction inwhich an analyte is converted into a product, the enzymes comprising afunctionalized FAD having a binding moiety which is chemicallyassociated with, attached to or chemically sorbed onto the surface ofthe electrode; (b) an electron mediator group which can transferelectrons between the surface of the electrode and the FAD: (c) anelectrical circuitry for charging the electrode and measuring theelectrical response.
 5. A system according to claim 4, wherein theelectron mediator group forms part of or is covalently bound to thefunctionalized FAD.
 6. A system according to claim 4, wherein theelectron mediator group is covalently bound to the enzyme.
 7. A systemaccording to claim 4, wherein the electron mediator group is freelytumbling in a medium surrounding the electrode.
 8. A method fordetermining the presence of an analyte in a liquid medium the methodcomprising: (a) providing a system according to claim 4, (b) introducinga sample of said liquid medium into the electrochemical cell of thesystem; (c) charging the electrode and measuring the electricalresponse, a change in the electrical response as compared to anelectrical response under the same condition in a control medium whichdoes not comprise the analyte, indicating the presence of the analyte inthe system.
 9. A method according to claim 8, wherein the analytedetermination is performed in vivo.
 10. An electro chemical system forthe recording of optical signals having a first wavelength andelectrical transduction of the recorded signals, the system having anelectrochemical cell comprising: (a) an electrode carrying immobilizedFAD-dependent redox enzymes, the enzyme having a functionalized FADcomprising a photoisomerizable group which changes its isomerizationstate from a first to a second state upon photostimulation of light ofthe first wavelength, a change in the isomerization state giving rise toa change in the rate of catalytic activity of the redox enzyme; (b) anelectron mediator group which can transfer electrons between theelectrode and the FAD; (c) a substrate for the catalytic activity of theenzyme; and (d) an electric circuitry for charging the electrode andmeasuring the electrical response.
 11. A system according to claim 10,wherein the enzyme is immobilized onto the surface of the electrodethrough a linking group which is covalently bound to an external moietyon the surface of the enzyme.
 12. A system according to claim 10 or 11,wherein the electron mediator group is freely tumbling in the mediumsurrounding the electrode.
 13. A system according to claim 10,comprising a light switch emitting light at a second wavelength, saidsecond wavelength photoisomerizes the photoisomerizable group from thesecond state to the first state.
 14. A method for recordal of opticalsignals having a first wavelength and electrical transduction of therecorded optical signals, the method comprising: (a) providing a systemaccording to claim 10: (b) exposing the electrode to a light source; (c)charging the electrode and measuring the electrical response, changingthe electrical response indicating exposure to light having said firstwavelength.